This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy of inherited diseases affecting the function of human phagocytic cells such as neutrophils,eosinophils, and monocytes. The scope of this project includes studies of the basic biology of human and animal blood stem cells; studies of the differentiation of PBHP into phagocytic cells; and the functional properties of mature phagocytic cells. During the past year we have continued to define conditions for optimum harvest, purification and culture of primitive human hematopoietic cells (CD34 positive cells). We determined optimum dosing of subcutaneous granulocyte colony stimulating factor (G-CSF) to recruit primitive CD34+ progenitor cells from bone marrow to peripheral blood, finding that G-CSF administered at 10 ug/kg subcutaneously daily for 6 days was best and that higher dosing increased side effects without enhancing mobilization. In other studies we demonstrated in mice that engraftment of congenic progenitor cells as a model of autologous transplantation could be enhanced by even low dose radiation and that at low radiation doses (30 cGy to 400 cGy) percent engraftment doubled with each 50 cGy increase. Murine retrovirus vectors used for clinical gene transfer require cell cycling (cell division) for optimum gene transfer. However, the most primitive progenitors (which are surface CD34 antigen high and CD38 surface antigen low) are mostly non-cycling. In current ongoing studies we have employed a novel fluorescence flow cytometry 4 color assay to demonstrate that primitive progenitors mobilized to peripheral blood have a higher percentage of cells in the G1, phase of the cell cycle than similar cells from bone marrow. As part of our studies of phagocytic cell host defense functions we have examined the structure of nitric oxide synthase - 2 (NOS-2). This enzyme produces nitric oxide which plays a role in host defense against intracellular pathogens like tuberculosis. We have created recombinant portions of this molecule which contain either the reductase activity (flavin and NADPH binding regions) or oxidant activity (heme binding region). Each of these regions is functional alone. This has allowed delineation of the role of cofactors in regulation of these functional activities. This may allow development of new drugs which affect NOS function.